Production of Recombinant Proteins in Plants

By Associate Professor of Biology Matt Pelletier

This past summer I spent five weeks working with Houghton College biology students Rebecca Dix (2009) and Hannah Stoveken (2009) on a research project aimed at using plants as “bioreactors” to produce therapeutically useful proteins. With the advent of recombinant DNA technology, one can theoretically take any cloned gene and transfer it to another host for production of proteins. Because plants are photosynthetic, produce a significant amount of biomass, and are easy to scale up in numbers, plants have significant potential advantages over other hosts for foreign protein production. This technology in theory produces an unlimited supply of human proteins for therapeutic use. For our project, we sought to express the blood-coagulation factor thrombin in Nicotiana benthamiana, a relative of tobacco.

During the summer, Hannah and Rebecca spent time synthesizing the necessary recombinant DNA molecules required for expression in plants. This involved several weeks of work that was successfully completed. The recombinant molecules were sequenced to verify no mutations occurred in the synthesis, and we attempted to transfer the molecules into Agrobacterium tumefaciens, a bacterium used in the transfer of the recombinant thrombin gene into the plant. After encountering several technical difficulties, we have since begun trying another method to transfer the molecules into the A. tumefaciens.

While we have not yet successfully expressed human thrombin in N. benthamiana, we decided to at least try production of another protein in the plants-one that is easy to detect and that others have already successfully produced in plants. This protein, called green fluorescent protein (GFP) was first identified in certain species of jellyfish. It is unique in that it will fluoresce a bright green color on exposure to ultraviolet light. To practice the method of production that we plan to use to make thrombin, Hannah and Rebecca infiltrated A. tumefaciens carrying the gene encoding GFP into the leaves of N. benthamiana plants. After 48 hours, a leaf from a leaf infiltrated with media only (negative control) or one infiltrated with A. tumefaciens was exposed to UV light and photographed. Expression of GFP was observed in the leaf infiltrated with A. tumefaciens, but not the leaf infiltrated with growth media only (Figure 1). We hope to conduct tests with the human thrombin gene as soon as the appropriate recombinant molecules are transferred into A. tumefaciens.

Hannah will be presenting our group’s work at the Rochester Academy of Science at the November 1 meeting.

Figure 1 Expression of GFP in Leaves of N. benthamiana:

The leaf on the left was infiltrated with media only while the leaf on the right was infiltrated with A. tumefaciens harboring the GFP gene.

After 48 hours, the leaves were removed, placed on a UV light box, and photographed.

Notice how the leaf on the right has fluorescent spots while the one on the left does not.